The endogenous polyamines, putrescine, spermine, and spermine contribute to many essential cellular functions through their interactions with DNA, RNA, proteins, and lipids (Pegg, A. E. Cancer Res. 48:759-774 (1988); Heby, O. et. al., Trends Biochem. Sci. 15:153-158 (1990); Jänne, J. et. al., Ann. Med. 23:241-259 (1991); Brooks, W. H. Med Hypotheses 44:331-338 (1995); Igarashi, K. et. al., Biochem. Biophys. Res. Commun. 271:559-564 (2000); Casero, R. A. et. al., J. Med. Chem. 44:1-26 (2001)). Polyamines are essential for cell proliferation through their involvement in DNA replication, cell cycle regulation, and protein synthesis. Depletion of intracellular polyamine levels inhibits cell growth. Antizyme regulates polyamine levels both by inhibiting polyamine biosynthesis and uptake/import. The importance of their function is highlighted by the fact that specific biosynthesis, degradation, uptake and excretion pathways tightly control cellular polyamine levels (Heby, O. Differentiation 19:1-20 (1981); Seiler, N. et. al., Int. J. Biochem. 22:211-218 (1990); Seiler, N. et. al., J. P. Int. J. Biochem. Cell Biol. 28:843-861 (1996)). Excessive cell growth has been correlated with high levels of intracellular polyamines (Pegg, A. E. Cancer Res. 48:759-774 (1988)). Numerous tumor cell types have been analyzed and shown to have higher polyamine levels than normal, non-tumorigenic cells. Within a single tumor type, the more highly malignant tumors often have higher polyamine levels (Kurihara, H. et. al., Neurosurgery 32:372-375 (1993)). For these reasons, depletion of intracellular polyamine levels is an attractive approach for the inhibition of uncontrolled or undesirable cell growth.
Ornithine decarboxylase (ODC) is the rate-limiting enzyme of cellular polyamine synthesis, converting ornithine to putrescine. Putrescine is then converted to both spermidine and spermine by the sequential transfer of an aminopropyl group from decarboxylated-S-adenosylmethionine. Increasing concentrations of intracellular polyamine levels induce the production of antizyme which negatively regulates ODC by binding to it and targeting it for destruction. Antizyme has also been shown to inhibit polyamine uptake (Mitchell, J. L. et. al., Biochem. J. 299:19-22 (1994); Suzuki, T. et. al., Proc. Natl. Acad. Sci. USA 91: 8930-8934 (1994); Sakata, K. et. al., Biochem. Biophys. Res. Commun 238:415-419 (1997)) and recent evidence suggests that antizyme may increase polyamine excretion (Sakata, K. et. al., Biochem J. 347:297-303 (2000)). Therefore, antizyme can very effectively limit the accumulation of cellular polyamines.
Antizyme has been found in vertebrates, fungi, nematodes, insects and eukaryotes (Ivanov, I. et. al., Nucleic Acids Res. 28:3185-3196 (2000)). Three antizyme isoforms, AZ1, AZ2 and AZ3, have now been identified among vertebrates. Both AZ1 and AZ2 have wide tissue distribution but AZ2 mRNA is less abundantly expressed. AZ3 is expressed only in the testis germ cells (Ivanov, I. et. al., Proc. Natl. Acad. Sci. USA 97: 4808-4813 (2000); Tosaka, Y. et. al., Genes to Cells 5:265-276 (2000)) where expression begins early in spermiogenesis and finishes in the late spermatid phase. Antizyme production is controlled by a unique regulatory mechanism known as translational frame shifting (Matsufuji, S. et. al., Cell 80: 51-60 (1995)). The antizyme gene consists of two overlapping open reading frames (ORFs). The bulk of the coding sequence is encompassed in the second (ORF2) but it does not contain an initiation codon. ORF1 is short but contains two AUG initiation codons. Either one of the initiation codons can be used to initiate translation but normally little full length mRNA is made unless a +1 frameshift occurs just before the ORF1 UGA stop codon enabling translation to continue. Only minute quantities of antizyme are generally present in mammalian tissues. Polyamines and agmatine have been found to greatly enhance the efficiency of frameshifting (Hayashi, S. et. al., Trends Biochem. Sci. 21:27-30 (1996); Satriano, J. et. al., J. Biol. Chem. 273:15313-15316 (1998)). Vertebrates possess three elements that control frameshifting, the UGA stop codon in ORF1, a stem-loop structure 3′ to the ORF1 UGA that can base pair with a portion of the loop and conserved sequence motifs within the 3′ region of ORF1 (Matsufuji, S. et. al., Cell 80: 51-60 (1995)). It is unclear how or if polyamines interact directly with these structural elements to induce frameshifting. It is possible that there are unknown mediators that may involve the ribosome.
ODC is enzymatically active only as a homodimer since the active site contains structural contributions from both monomers. The interaction between the monomers is weak; whereas, antizyme has a high affinity for the ODC monomer. Antizyme binding disrupts the homodimer interface leading to the formation of two antizyme-ODC heterodimers that are now enzymatically inactive (Kameji, T. et. al., Biochim. Biophys. Acta 717:111-117 (1982); Kern, A. D. et. al., Struct. Fold. Des. 7:567-581 (1999)). Antizyme directs the ODC monomer to the proteosome where it is degraded without ubiquitination (Murakami, Y. et. al., Nature 360:597-599 (1992); Tokunaga, F. et. al., J. Biol. Chem. 269:17382-17385 (1994)). Antizyme is then released and free to interact with and destroy additional ODC monomers in a catalytic fashion. The AZ2 isoform has not been shown to catalytically induce the degradation of ODC, although AZ2 has been shown to inhibit both ODC and polyamine uptake equipotently (Zhu, C. et. al., J. Biol. Chem. 274: 26425-26430 (1999). AZ3 is the most recently discovered antizyme and has also been shown to inhibit ODC (Ivanov, I. et. al., Proc. Natl. Acad. Sci. USA 97:4808-4813 (2000); Tosaka, Y. et. al., Genes to Cells 5:265-276 (2000)).
Antizyme is regulated by antizyme inhibitor, which has a higher affinity towards antizyme than ODC (Fujita, K. et. al., J. Biol. Chem. 274:26424-26430 (1982); Kitani, T. et. al., Biochim. Biophys. Acta 991:44-49 (1989); Murakami, Y. et. al., Biochem. J. 259:839-845 (1989)). Thus it may rescue ODC from degradation by displacing it from antizyme. Antizyme inhibitor, like ODC, forms a homodimer and has a high degree of sequence homology with ODC. However, it does not form heterodimers with ODC (Murakami, Y. et. al. J. Biol. Chem. 271:3340-3342 (1996)) and lacks ODC activity. Antizyme inhibitor has been shown to be rapidly induced in growth-stimulated fibroblasts and release ODC from antizyme suppression (Nilsson, J. et. al., Biochem. J. 346:699-704 (2000)).
Frameshifting can be detected using a dual luciferase reporter system that measures the efficiency of antizyme translational frameshifting (Grentzmann, G. et. al, RNA 4:479-486 (1998); Howard, M. et. al., Genes to Cells 6:931-941 (2001)). Frameshifting efficiency is determined by comparing the ratio of firefly luciferase to renilla luciferase activity in cells transfected in parallel using a control vector containing a constitutive +1 frameshift (AZ-IF) that measures the in-frame translation efficiency and a vector containing the inducible 0 to +1 frameshift (AZ1) construct. In these constructs, the renilla luciferase gene is upstream of the firefly luciferase gene which are separated by a short cloning sequence containing the portions of antizyme 1 and 2 known to contain the mRNA signals for polyamine stimulated frameshifting. Using a 96-well format, this assay system gives a quantitative measure of the efficiency of the polyamines, polyamine analogs and other compounds to induce frameshifting in a cell-based bioassay. Cells must be pretreated with α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, prior to screening to decrease the basal antizyme frameshifting levels and increase the sensitivity to polyamine or compound-mediated stimulation of antizyme frameshifting.
In one of the first systematic assessments of antizyme induction by polyamine analogs, oligoamines such as octamines, decamines and dodecamines were found to induce antizyme to varying degrees (Mitchell, J. L. A. et. al., Biochem. J. Vol. 366, p. 663-671, 2002). These levels correlated with the cellular levels of antizyme as measured by Western blotting. The differences in the levels of antizyme appeared to be a result of dissimilar rates of protein synthesis since the half-life of antizyme (T½˜75 min.) did not appear to be controlled by the polyamine analog. Therefore, it is presumable that the analogs have varying abilities to stimulate the +1 translational frameshift. A number of compounds such as bisethylnorspermine, bisethylhomospermine and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) were found to induce antizyme as well as spermine. However, certain conformational restrictions within the polyamine analogs such as three, four and five-membered rings or triple bonds between the central nitrogens negatively affected antizyme induction. Many of the oligoamines greatly exceeded spermine in their ability to induce antizyme (super-induction) when tested at the same concentration (10 μM). The amount of antizyme frameshifting was found to correlate with the degree of growth inhibition. The oligoamines induced immediate cessation of cell growth, which was speculated to result from the super-induction of frameshifting. However, the authors also noted that these compounds might have other mechanisms of action leading to their observed cytotoxicity.
It is plausible that some antizyme inducers will also directly inhibit the enzymatic activity of ODC. A number of putrescine analogs have been found to be potent reversible inhibitors of ODC. For example, 1,4-diamino-trans-2-butene inhibits ODC with a K1 of 2 μM and 1,4-phenylenediamine somewhat less potently inhibits ODC with a K1 of 46 μM (Relyea, N. et. al., Biochem. Biophys. Res. Comm. 67:392-402 (1975); Solano, F. et. al., Int. J. Biochem. 20:463-470 (1988). Compounds of this nature may enhance polyamine depletion because ODC is inhibited in both a direct and indirect manner through induction of antizyme.
Polyamines may arrest prostate cell growth in the G1 phase by inducing antizyme. The prostate is the only vertebrate organ that synthesizes polyamines for export. As such, this tissue is exposed to higher concentrations of the polyamines. Spermine has been found to be a naturally occurring inhibitor of prostatic carcinoma cell growth in vitro and in vivo (Smith, R. C. et. al., Nature Med. 1:1040-1045 (1995)). Subsequently, it was found that spermine could induce G1 arrest in poorly metastatic prostatic carcinomas but not in highly malignant cells (Koike, C et. al., Cancer Res. 59:6109-6112, (1999)). Furthermore, antizyme could be induced only in the poorly metastatic prostatic carcinomas. Antizyme was later found to affect the cell-cycle of prostatic carcinoma cells with the discovery that it could interact with G1 cyclin D1 and its associated cyclin-dependent kinase, cdk4 (Coffino, P. Nat. Rev. Mol. Cell. Biol. 2:188-194 (2001)). The degradation of cdk4 and cyclin D1 were dependent on antizyme and independent of ubiquitin using in vitro purified proteasomes. The steady-state levels of the cyclin and kinase decreased when the polyamine levels were experimentally raised in the cultured cells. It has been proposed that prostatic cells that lose the ability to activate antizyme may eventually become malignant (Koike, C et. al., Cancer Res. 59:6109-6112, (1999)).
A number of studies have looked at both transient and inducible overexpression of antizyme in cell lines and animal models. Anti-tumor activity was shown in a study by Iwata and colleagues (Iwata, S. et. al., Oncogene 18:165-172 (1999)) using ectopically expressed inducible antizyme. In this study, nude mice were inoculated with H-ras transformed NIH3T3 cells expressing an inducible antizyme vector. Induction of antizyme blocked tumor formation in these mice and induced cell death in vitro. Intracellular polyamine levels were also measured. Both putrescine and spermine were completely depleted within 12 hours of induction. Spermine was also significantly reduced but over a slower time frame. Some of these observations were verified in another report that used a glucocorticoid (dexamethasone)-inducible promoter to force expression of antizyme in HZ7 cells (Murakami, Y. et. al., Biochem. J. 304:183-187 (1994)). Dexamethasone inhibited growth of this cell line, depleted putrescine levels, severely decreased spermidine levels but did not affect spermine levels. Addition of exogenous putrescine restored the intracellular putrescine levels and partially restored spermidine levels. In a third study, Tsuji and colleagues (Tsuji, T. et. al., Oncogene 20:24-33 (2001)) developed a hamster malignant oral keratinocyte (HCPC-1) cell line that stably expressed antizyme. Ectopic expression of antizyme suppressed tumor mass in nude mice by about 50%. In vitro, ectopic expression significantly increased the doubling time of antizyme transfectants and the antizyme transfectants demonstrated significantly less growth in soft agar. There was also a substantial increase in G1 phase cells with a corresponding decrease in S phase cells. These cells also showed morphological alterations suggesting terminal differentiation. This was accompanied by an increase in demethylation of DNA CCGG sites of 5-methyl cytosines. It was proposed that antizyme mediates a novel mechanism in tumor suppression by reactivating key cellular genes silenced by DNA hypermethylation during cancer development. In yet another example, transgenic mice that overexpress ODC in keratinocytes have been shown to develop a high rate of spontaneous and induced skin cancer (Megosh, L. et. al., Cancer Res. 55:4205-4209 (1995)). A reduction in the frequency of induced skin-tumors was observed in the skin of these transgenic mice expressing antizyme (Feith, D. et. al. Cancer Res. 61:6073-6081 (2001)).
Polyamines have been found to play a central role in hair follicle cell growth, a highly proliferative tissue, with a cell turnover time of between 18-23 hours. ODC plays a functional role in hair follicle growth, which is characterized by cyclic transformations from active growth and hair fiber production (anagen) through regression (catagen) into a resting phase (telogen). In mice, ODC is expressed in ectodermal cells at sites where hair follicles develop during embryonic development (Nancarrow, M. J., et. al., Mech. Dev. 84: 161-164 (1999); Schweizer, J. In: Molecular Biology of the Skin: The Keratinocyte, Darmon M, et. al., Eds., Academic Press, New York, 1993, pp 33-78). In proliferating bulb cells of anagen follicles, ODC is abundantly expressed except for a pocket of cells at its base. ODC protein expression is down regulated when the hair follicle enters catagen and is not detected in telogen. ODC protein expression does not resume until new follicle formation commences. A more complex expression of ODC is found in vibrissae (beard hair). ODC is expressed in the keratinocytes of the vibrissal hair shaft as well as in the bulb and outer root sheath cells near the follicle bulge. In comparison, ODC expression is very low in interfollicular epidermis.
Numerous studies have shown that inhibition of ODC with DMFO, an irreversible inhibitor of ODC, reduces hair growth in mammals. Mice were found to have reduced hair growth when DFMO was systemically delivered via the drinking water (Takigawa, M. et. al., Cancer Res. 43:3732-3738 (1983)). Intravenous administration of DFMO decreased wool growth in sheep (Hynd, P. I. et. al., J. Invest. Dermatol. 106:249-253 (1996)) and oral administration of DFMO in cats and dogs produced alopecia and dermatitis (Crowell, J. A. et. al., Fundam. Appl. Toxicol. 22:341-354 (1994)). Additional evidence that ODC plays a role in hair follicle regulation resulted from a study in humans that were being treated for acute Trypanosoma brucei infections (African sleeping sickness) (Pepin, J. et. al. Lancet 2:1431-1433 (1987)) using DFMO. Patients using this treatment showed signs of hair loss mainly on the scalp but it was reversible after discontinuing treatment.
The development of a number of transgenic mice either overexpressing spermidine/spermine N1-acetytransferase (SSAT) or ODC have contributed additional evidence that distorted tissue polyamine pools leads to hair loss (Pietilä, M. et. al., J. Biol. Chem. 272:18746-18751 (1997); Suppola, S. et. al., Biochemistry 7338:311-316 (1999); Megosh, L. et. al., Cancer Res. 55:4205-4209 (1995)). SSAT is a key enzyme in the catabolism of polyamines that is rate-limiting for the conversion of spermine to spermidine and spermidine to putrescine. Both transgenic animal models showed permanent hair loss in which the normal hair follicles were transformed into dermal cysts that progressively increased in size as the animals aged (Pietilä, M. et. al., J. Biol. Chem. 272:18746-18751 (1997); Suppola, S. et. al., Biochemistry 7338:311-316 (1999); Soler, A. P. et. al., J. Invest. Dermatol. 106, 1108-1113 (1996); Megosh, L. et. al., Cancer Res. 55:4205-4209 (1995)). This was manifest as a thickening and excessive skin folding of the epidermis. The common phenotypic feature that each of these animal models shared was a massive over accumulation of putrescine in the skin (Pietila, M. et. al., J. Invest. Dermatol. 116:801-805 (2001)). It was proposed that elevated levels of polyamines and especially putrescine favor continuous proliferation of epithelial cells leading to the formation of follicular cysts and hair loss. Low levels of putrescine favor differentiation of the outer root sheath keratinocytes and are not permissive for proliferation.
Polyamine biosynthesis has also been shown to be essential during the activation of immunocompetent cells (Fillingame, R. H. et. al., Proc.Natl.Acad.Sci. USA 72:4042-4045, (1975); Korpela, H. et. al., Biochem.J. 196:733-738 (1981)). Studies with DFMO confirm that polyamine depletion therapy can inhibit the immune response and may be a successful therapy against a number of autoimmune diseases. Both humoral and cell-mediated immune responses were affected by the anti-proliferative effect of polyamine depletion. DFMO treatment of mice challenged with tumor allografts resulted in modified cytotoxic T-lymphocyte and antibody responses (Ehrke, J. M. et. al., Cancer Res. 46:2798-2803 (1986)). Reports by Singh et al. indicate that DFMO treatment may also ameliorate acute lethal graft versus host (ALGVH) disease in mice (Singh, A. B. et. al., Clin.Immunol. Immunopathol. 65:242-246 (1992)). Murine ALGVH represents a model of human GVH that contributes to the morbidity and mortality of bone marrow transplantation in humans and is characterized by anemia and the loss of T cell function and numbers. In this study, treatment of ALGVH mice with DFMO decreased mortality and anemia while preserving the cytotoxic T cell and natural killer cell population of the host. Polyamine depletion therapy using DFMO has also been shown to benefit lupus-prone female NZB/W mice (Thomas, T. J. et. al., J.Rheumatol. 18:215-222 (1991)). Anti-DNA antibody production, immunoglobulin G and A synthesis, proteinuria and blood urea nitrogen were significantly reduced in treated mice.
Chemotherapeutics and radiation therapies target rapidly dividing cancer cells but they inadvertently affect the rapidly dividing epithelial cells of the mouth and intestine, hair follicles and hematopoietic cells in bone marrow. If the epithelial cells of the mouth or intestine become damaged and depleted, thinning and ulceration can result (mucositis) leading to pain and potential infection. Oral mucositis is also the result of damaged stem cells. Oral tissues are particularly painful if damaged.
Under normal conditions, the lining of the intestine is continuously being renewed through the proliferation of epithelial stem cells and their progeny in the crypts of villi (Booth, D, et. al., J. Natl Cancer Inst Monogr 29:16-20 (2001)). When damage occurs (e.g., radiation or cytotoxic insult), a burst of proliferation/regeneration occurs in undamaged stem cells. A number of proposals to limit the damage to stem cells and enhance regeneration have been made. One strategy has been to arrest the cell cycle progression and accumulate cells in G0 or G1 during radiation or chemotherapy treatment to make them more resistant to damage. Other strategies include increasing the number of stem cells prior to potential damage or enhancing proliferation after damage (Farrell, C. L. et. al., Cancer Res. 58: 933-939 (1998)). Polyamines are taken up from the gut by normal and neoplastic epithelial cells of the gut mucosa, especially during periods of cell proliferation (Milovic V. et. al., Eur J Gastroenterol Hepatol. 13:1021-5 (2001)). The involvement of polyamines in proliferation of intestinal epithelial cells has been demonstrated using the nontransformed small intestinal cell line from rats, IEC-6, where polyamines increased DNA synthesis (Olaya, J. et. al. In Vitro Cell Dev Biol. Anim. 35:43-8, (1999)). The chemotherapeutic agent camptothecin, a DNA topoisomerase I inhibitor, can induce apoptosis in IEC-6 cells. However, reducing polyamines can have a protective effect. When IEC-6 cellular polyamines were reduced with DFMO, apoptosis due to camptothecin was delayed (Ray, R. M. et. al., Am J Physiol Cell Physiol 278:C480-489 (2000)). This may be due to G1 cell cycle arrest, which has been demonstrated to occur in IEC-6 cells incubated with DFMO (Ray R. M. et. al. Am. J. Physiol. 276:C684-91 (1999)). A more efficient depletion of polyamines with synthesis and uptake inhibition through induction of antizyme could provide significant protection against mucositis after radiation or chemotherapy.